Theregulatoryprocessknownasproteinubiquitinationmodifiescellularproteinswith far-reachingimpactsonhumanhealthanddisease.Itisinvolvedineveryknown biologicalprocessandisimplicatedinagrowingrangeofdiseasesthatincludes cancers,neurodegenerativediseases,musclewasting,etc.Studiesconducted worldwideoverthepasttwentyyearshavedefinedthemechanismsbywhichprotein ubiquitinationiscarriedout,withoneobviousexception.Almostlittleisknownabouthow substrates(i.e.,thecellularproteinsthataremodifiedbythissystem)arerecognized andhowtheyfitintotheultimatemechanism.Inthecominggrantperiod,weproposeto rectifythissituationfortwoimportantubiquitinE3ligases,BRCA1/BARD1andHHARI. Ourgoalsaremadepossiblebyseveralimportantadvances,includingdefinitionofbona fidesubstratesforeachoftheseE3sandtheabilitytochemicallysynthesize biochemicallyhomogeneousversionoftheproducts.Wewillalsoinvestigatetwo uncharacterizedhumanE2Ubiquitin-conjugatingenzymesthateachhave unusual/uniqueactivitiesinthattheyattachUbtoproteingroupsotherthanlysines, therebyexpandingtheuniverseofpossibleattachmentsites.Wewilluseacombination ofbiochemical,structural,andcellularapproachestoaddressquestionsoffundamental importanceandgeneralrelevance.